Methods for treating brain injury

ABSTRACT

The treatment options for treating blast-induced and noise-induced traumatic brain injury and tinnitus are limited. Thus, the current invention provides methods for treating traumatic brain injury and tinnitus. The methods involve administering a pharmaceutically effective amount of a composition comprising 2,4-disulfonyl α-phenyl tertiary butyl nitrone and N-acetylcysteine (NAC).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/046,676, filed Feb. 18, 2016, which is a continuation of U.S. patentapplication Ser. No. 13/983,515, filed Aug. 2, 2013, now U.S. Pat. No.9,289,404, which is the National Stage of International PatentApplication No. PCT/US2012/023855, filed Feb. 3, 2012, and published asWO 2012/106654, which claims priority from U.S. Provisional PatentApplication No. 61/439,671, filed Feb. 4, 2011. The contents of theseapplications are incorporated herein by reference in their entirety.

BACKGROUND

There is increased evidence that blast overpressure is transmittedacross the skull into the brain. This sets up the potential to causetraumatic brain injury (TBI) including damage to the central auditorycenters of the brain, e.g. brainstem, temporal lobe, and thalamus whichcould explain symptoms such as hearing loss, dizziness, and tinnitus. Ofparticular significance is the observation that blast-related TBIproduces significantly greater rates of hearing loss and tinnitus (60%)compared with non-blast related TBI. Similarly, intense sound- ornoise-induced changes in the central auditory structure have beenreported, including the cochlear nucleus, inferior colliculus, medialgeniculate body and primary auditory cortex.

Although, some mechanical damage will have permanent effects, much ofthe long-term damage results from secondary molecular and cellularprocesses that are triggered by the blast-induced trauma amplify theeffects of mechanical damage. TBI initiates an almost immediate injuryprocess including contusion, diffuse axonal injury, hematoma,subarachnoid hemorrhage followed shortly thereafter by a variety ofsecondary injuries. The secondary injuries can include ischemia, edema,oxidative damage, decreased ATP, cytoskeleton changes, inflammation, andactivation of cell death pathways. To date, an effective therapeuticapproach that addresses these secondary molecular and cellular processeshas yet to be thoroughly investigated. Thus, a substantial need existsfor treatment methods and compounds suitable for treating these issuesassociated with victims of TBI.

SUMMARY

A method for treating noise-induced and blast-induced traumatic braininjury is provided. In one embodiment, a pharmaceutically effectiveamount of a composition comprising 2,4-disulfonyl α-phenyl tertiarybutyl nitrone is administered to an organism that has sufferednoise-induced or blast-induced brain injury. In one aspect of thecurrent embodiment, the composition further comprises N-acetylcysteine(NAC). In another aspect of the current embodiment, the composition isadministered orally.

A method for treating traumatic brain injury is provided. In oneembodiment, a pharmaceutically effective amount of a compositioncomprising 2,4-disulfonyl α-phenyl tertiary butyl nitrone isadministered to an organism that has suffered traumatic brain injury. Inone aspect of the current embodiment, the composition further comprisesN-acetylcysteine (NAC). In another aspect of the current embodiment, thecomposition is administered orally.

In another embodiment, the method for treating brain injury comprisesadministering a composition to an organism that has suffered a braininjury, wherein the composition comprises 4-hydroxy-α-phenyl butylnitrone. In one aspect of this embodiment, the composition furthercomprises NAC. In another aspect of the current embodiment, thecomposition further comprises NAC and Acetyl-L-Carnitine (ALCAR). In yetanother aspect of the current embodiment, the composition isadministered orally.

A method for treating tinnitus is provided. In one embodiment, apharmaceutically effective amount of a composition comprising2,4-disulfonyl α-phenyl tertiary butyl nitrone is administered to anorganism that is suffering from noise-induced tinnitus. In one aspect ofthe current embodiment, the composition further comprisesN-acetylcysteine (NAC). In another aspect of the current embodiment, thecomposition is administered orally.

In another embodiment, the method for treating noise-induced tinnituscomprises administering a composition to an organism that is sufferingfrom noise-induced tinnitus, wherein the composition comprises4-hydroxy-α-phenyl butyl nitrone. In one aspect of this embodiment, thecomposition further comprises NAC. In another aspect of the currentembodiment, the composition further comprises NAC and Acetyl-L-Carnitine(ALCAR). In yet another aspect of the current embodiment, thecomposition is administered orally.

The current invention also relates to a method for increasing thebioavailability of a compound in the central nervous system. The methodinvolves administering 2,4-disulfonyl α-phenyl tertiary butyl nitrone toan organism in an amount sufficient to increase blood-brain barrierpermeability and administering the compound to the organism eitherconcurrently or following administration of 2,4-disulfonyl α-phenyltertiary butyl nitrone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F provide images of the dorsal cochlear nucleus (DCN)immunostained for c-fos in control subjects (1A), subjects exposed tonoise (1B—1 hour post noise; 1C—8 hours post noise; 1E—24 hours postnoise), and subjects treated with 2,4-disulfonyl α-phenyl tertiary butylnitrone (2,4-disulfonyl PBN) and NAC 4 hours after noise exposure (1D—8hours post noise; 1F—24 hours post noise).

FIG. 1G provides a bar graph representing the number of c-fos positivecells counted in the DCN from the images of FIG. 1A-1F, where NC=normalcontrols, H-N=hours post noise exposure, D-N=days post noise exposure,and T=received treatment of 2,4-disulfonyl PBN+NAC.

FIG. 2 provides a bar graph representing the number of c-fos positivecells in the posteroventral cochlear nucleus (PVCN) at various timepoints post noise exposure in the absence and presence of treatment with2,4-disulfonyl PBN) and NAC, where NC=normal controls, H-N=hours postnoise exposure, D-N=days post noise exposure, and T=received treatmentof 2,4-disulfonyl PBN+NAC.

FIGS. 3A-3F provide images of the anteroventral cochlear nucleus. (AVCN)immunostained for c-fos in control subjects (3A), subjects exposed tonoise (3B—1 hour post noise; 3C—8 hours post noise; 3E—24 hours postnoise), and subjects treated with 2,4-disulfonyl PBN and NAC 4 hoursafter noise exposure (3D—8 hours post noise; 3F—24 hours post noise).

FIG. 3G provides a bar graph representing the number of c-fos positivecells counted in the AVCN from the images of FIG. 3A-3F, where NC=normalcontrols, H-N=hours post noise exposure, D-N=days post noise exposure,and T=received treatment of 2,4-disulfonyl PBN+NAC.

FIG. 4 provides a bar graph representing a comparison of the resultsfrom FIG. 1G, FIG. 2 and FIG. 3G.

FIGS. 5A-5C provide images of the middle region of the DCN immunostainedfor precerebellin in control subjects (5A), subjects exposed to noise(5B), and subjects exposed noise and treatment with 4-hydroxy-α-phenylbutyl nitrone (4-OHPBN)+NAC+ALCAR (5C).

FIG. 5D provides a bar graph representing the density of precerebellinpositive cells in the lateral, middle, and medial regions of the DCN incontrol subjects, subjects exposed to noise, and subjects exposed noiseand treatment with 4-OHPBN+NAC+ALCAR.

FIGS. 6A-6C provide images of the middle region of the DCN immunostainedfor PEP-19 in control subjects (6A), subjects exposed to noise (6B), andsubjects exposed noise and treatment (4-OHPBN+NAC+ALCAR) (6C).

FIG. 6D provides a bar graph representing the density of PEP-19 positivecells in the lateral, middle, and medial regions of the DCN in controlsubjects, subjects exposed to noise, and subjects exposed noise andtreatment (4-OHPBN+NAC+ALCAR).

FIGS. 7A-7C provide images of the middle region of the DCN immunostainedfor NeuN in control subjects (7A), subjects exposed to noise (7B), andsubjects exposed noise and treatment (4-OHPBN+NAC+ALCAR) (7C).

FIG. 7D provides a bar graph representing the density of Neu-N positivecells in the lateral, middle, and medial regions of the DCN in controlsubjects, subjects exposed to noise, and subjects exposed noise andtreatment (4-OHPBN+NAC+ALCAR).

FIGS. 8A-8C provide transmission electron microscopic images of nerveterminals surrounding cartwheel cell bodies in the middle region of theDCN of control subjects (8A), subjects following noise exposure (8B) andsubjects treated with 4-OHPBN+NAC+ALCAR four hours following noiseexposure (8C).

FIGS. 8D-8F represent FIGS. 8A-8C, respectively, at a highermagnification.

FIGS. 8G-8I provide transmission electron microscopic images of nerveterminals surrounding the primary dendrites of cartwheel cell bodies inthe middle region of the DCN of control subjects (8G), subjectsfollowing noise exposure (8H) and subjects treated with4-OHPBN+NAC+ALCAR four hours following noise exposure (8I).

FIGS. 8J-8L, represent FIGS. 8G-8I, respectively, at a highermagnification.

FIG. 9 provides a bar graph representing the density of cells positivefor amyloid precursor protein (APP) in the hippocampus of subjectsexposed to blast, where NC=normal controls (no blast), H-B=hours postblast, D-B=days post blast, and T=treatment with 2,4-disulfonylPBN+NAC+4 hours post blast.

FIG. 10A provides a light microscopy image (4× magnification) DCNimmunostained for glial fibrillary acidic protein (GFAP) in a normalcontrol subject.

FIGS. 10B-10D provide light microscopy images (20× magnification) of themiddle region of the DCN immunostained for GFAP of normal controlsubjects (10B), subjects exposed to blast (10C), and subjects exposed toblast followed by treatment with 2,4-disulfonyl PBN+NAC (10D).

FIG. 10E provides a bar graph representing the density of GFAP positivecells in the lateral, middle, and medial regions of the DCN in controlsubjects, subjects exposed to blast, and subjects exposed to blastfollowed by treatment with 2,4-disulfonyl PBN+NAC.

FIG. 11 provides a bar graph representing the percentage increase inblood-brain barrier permeability in subjects exposed to blast in theabsence (control) and presence of 2,4-disulfonyl PBN (HPN-07)+NACadministered one hour post-blast.

FIGS. 12A-12C represent doublecortin immunostained sections of thehippocampus in control subjects not exposed to noise trauma (FIG. 12A),subjects 6 months post noise exposure without treatment (FIG. 12B) andwith HPN-07 treatment (FIG. 12C).

FIGS. 13A-13E represent doublecortin immunostained sections of theentorhinal cortex in control subjects note exposed to noise trauma (FIG.13A), subjects 21 days and 6 months post noise exposure withouttreatment (FIGS. 13B and 13D, respectively), and with HPN-07 treatmentat the same time points (FIG. 13G and 13E, respectively).

DETAILED DESCRIPTION

The invention provides methods for treating noise-induced tinnitus andtraumatic brain injury as well as brain injury related to oxidativestress, programmed cell death, or inflammatory processes. Traumaticbrain injury (TBI) is an alteration in brain function, or other evidenceof brain pathology, caused by an external force. The external forcescausing the TBI include, but are not limited penetrating injuries(penetration of an object into the brain) and non-penetrating or closedinjuries such as exposure to blasts or pressure (blast-induced TBI),head being struck by an object, or from exposure to noise (noise-inducedTBI) The current invention demonstrates the functionality of2,4-disulfonyl α-phenyl tertiary butyl nitrone and the synergisticeffect of combining the 2,4-disulfonyl α-phenyl tertiary butyl nitronewith N-acetylcysteine (NAC) in the treatment of tinnitus and traumaticbrain injury. For the purposes of the remainder of this disclosure,2,4-disulfonyl α-phenyl tertiary butyl nitrone will be referred to as2,4-disulfonyl PBN or HPN-07.

The 2,4-disulfonyl PBN has the following structure:

The acid form of the compound has the following structure:

The acid form may be a solid or found in low pH solutions. The ionizedsalt form of the compound exists at higher pH and may be represented byeither of the following structures:

In the salt form, X is a pharmaceutically acceptable cation. Mostcommonly, this cation is a monovalent material such as sodium, potassiumor ammonium, but it can also be a multivalent alone or cation incombination with a pharmaceutically acceptable monovalent anion, forexample calcium with a chloride, bromide, iodide, hydroxyl, nitrate,sulfonate, acetate, tartrate, oxalate, succinate, palmoate or the likeanion; magnesium with such anions; zinc with such anions or the like.Among these materials, the free acid and the simple sodium, potassium orammonium salts are most preferred with the calcium and magnesium saltsalso being preferred but somewhat less so. The 2,4-disulfonyl PBNcompound is described in detail by U.S. Pat. No. 5,488,145. The entiredisclosure of U.S. Pat. No. 5,488,145 is incorporated herein byreference. The salts of 2,4-disulfonyl PBN may also be used for thetreatment of brain injury and tinnitus in a manner similar to the use of2,4-disulfonyl PBN as discussed below.

Additionally, antioxidant peptides, which target the mitochondria, areuseful in the present invention and may be included as part of thecomposition for treating tinnitus and traumatic brain injury. Thesecompounds preclude the generation of intracellular reactive oxygenspecies (ROS) which leads to oxidative stress and damage of themitochondria. Oxidative damage of the mitochondria is known to causeapoptosis and necrosis leading to cell death. The preferred antioxidantpeptides are Szeto-Schiller (SS) peptides and their functional analogs.These compounds have alternating aromatic residues and basic aminoacids. In particular, peptides having tyrosine (Tyr) or dimethyltyrosine(Dmt) analogs can scavenge oxyradicals. These compounds inhibitoxidation of low-density lipoproteins. SS-peptides include compoundssuch as SS-31 (D-Arg-Dmt-Lys-Phc-NH₂) and SS-02 (Dmt-D-Arg-Phe-Lys-NH₂).In addition to the Tyr and Dmt containing SS-peptides, tryptophancontaining SS-peptides are also useful in the current invention.Finally, the amino acids found in the SS-peptides may be L or D and maybe naturally occuring, non-naturally occuring and derivatives ofnaturally occuring amino acids. In particular, the SS-peptides disclosedin PCT published application WO 2005/072295 are suitable for use in thecurrent invention. The entire disclosure of WO 2005/072295, published onAug. 11, 2005 is incorporated herein by reference. The composition ofthe current invention may optionally include antioxidant compoundsincluding, but not limited to, Acetyl-L-Carnitine (ALCAR), glutathionemonoethylester, ebselen, D-methionine.

In another embodiment, the current invention utilizes 4-hydroxy-α-phenylbutyl nitrone (4-OHPBN) or a derivative of 4-OHPBN alone or incombination with at least one antioxidant to treat brain injury andnoise-induced tinnitus. Additionally, the derivatives of the 4-OHPBN maybe formulated to enhance oral absorbtion, alter bioavailabilitykinetics, and/or formulated in a combination with one or more of theabove compounds. Preferably, the compositions for treating brain injuryand tinnitus will be administered orally. However, other methods whichdeliver the compositions systemically to the body should work equallywell.

As demonstrated in the Examples, the inventors have discovered that4-hydroxy-α-phenyl butyl nitrone (4-OHPBN) or 2,4-disulfonyl PBN,administered in combination with N-acetylcysteine (NAC) one to fourhours after noise exposure or blast exposure can prevent molecularchanges associated with the resulting traumatic brain injury.Furthermore, the areas of the brain affected by the noise or blastexposure, such as the dorsal cochlear nucleus, have been associated withthe etiology of tinnitus thereby providing a novel therapeutic approachto treatment of tinnitus.

Although the preferred embodiments of the current invention aredescribed primarily with respect to noise-induced tinnitus andnoise-induced and blast-induced traumatic brain injury, it should beappreciated that the methods and compositions described herein may beused to treat traumatic brain injury and tinnitus caused by a variety ofdifferent events or factors. The methods described herein may bespecifically useful in treating secondary injuries resulting from TBIincluding ischemia, edema, oxidative damage, decreased ATP, cytoskeletonchanges, inflammation, and activation of cell death pathways.

The compositions of the current invention will preferably beadministered orally; however, other delivery methods including, but notlimited to, intravenously, subcutaneously, by inhalation, sublingually,subdermally or intrathecally. Further the active composition may beadministered as a nanoparticle or dendrimer formulation. Thenanoparticle may be multifunctional and composed of a polymer andparamagnetic iron oxide particles to allow the application of externalmagnetic forces to aid in the delivery of the drug to the desired targetsuch as the dorsal cochlear nucleus. Additionally, the composition maybe formulated with additives known to those skilled in the art toenhance oral absorbtion and alter bioavailability kinetics.

EXAMPLES

In the examples to follow, the inventors demonstrate that exposure tohigh levels of noise and blasts (high pressure exposure) can inducemolecular and cellular changes associated with traumatic brain injury inthe certain regions including the dorsal cochlear nucleus, thehippocampus, and the entorhinal cortex. Moreover, the inventorsdemonstrate that administration of a composition comprising2,4-disulfonyl PBN (HPN-07) either alone or in combination with NAC canreverse the cellular changes associated with the traumatic brain injury.In doing so, symptoms of the traumatic brain injury, includingnoise-induced tinnitus can be lessened. Alternatively, the inventorsdemonstrate that a composition comprising 4-hydroxy-α-phenyl butylnitrone, NAC and ALCAR can have similar therapeutic effects.

Example 1

The purpose of this example is to demonstrate that a compositioncomprising 2,4-disulfonyl PBN (HPN-07) and NAC is effective in reversingmolecular changes indicative of noise-induced traumatic brain injury inthe dorsal cochlear nucleus.

MRI studies of animals experiencing chronic acoustic exposure-inducedtinnitus have demonstrated increased brain activity in the dorsalcochlear nucleus. Expression of c-fos, an immediate-early gene, iswidely used as an accepted marker of neuronal activity. Increases inc-fos expression have been observed in the central nerve system fewhours to 5.5 weeks after noise exposure suggesting that c-fos expressionmight represent a neural correlate of tinnitus or of plasticityassociated with noise induced tinnitus. Increased expression of c-foshas also been associated with brain injury. in the present example,c-fos expression was examined 1 hours to 21 days following 115 dB SPLoctave-hand noise exposure both with and without administration of acomposition comprising HPN-07 and NAC.

Adult rats (Spraue Dawley, 4-6 in each group) were used in the study.The animals in the noise groups (N) and the noise plus treatment groups(N/T) were exposed to 115 dB SPL octave-band noise centered at 14 kHzfor 1 hour. A composition comprising 20 mg/kg HPN07 and 50 mg/kg NAC wasintraperitoneally administrated 4 hours after the noise exposure andtwice a day for the next 2 days. Rats receiving no noise exposure servedas normal controls (NC). Auditory brainstem response (ABR) anddistortion product otoacoustic emission (DPOAE) were recorded beforenoise exposure and euthanasia. Brainstems were harvested 1 hour (1H),8H, 24H, 7 days (7D) and 21D after noise exposure and processed forparaffin embedding and sectioning at a thickness of 6 μm. Cells positivefor c-fos were identified in sections by immunohistochemical staining.Immuno-density was determined by light microscopy as the number ofpositive cells per sq. mm. The data was analyzed statistically (onewayANOVA and Tukey HSD tests).

FIG. 1. depicts examples of c-fos immunostaining images obtained fromthe dorsal cochlear nucleus (DCN) of the normal control (A), the noiseexposure (B, C, E) and the noise/treatment (D, F) groups by lightmicroscopy. Numerous positive c-fos stained cells were mainly found inthe fusiform soma layer with few positive cells in the molecular anddeep layers 1H after noise exposure (B), suggesting that c-fosexpression was up-regulated in the DCN immediately after noise exposure.The number of positive cell decreased 8H (C) and 24 H (E), and returnedto the normal lever 7D and 21D (not shown) after noise exposure.Positive stained cells in the DCN were counted and statisticallyanalyzed (G). A significant increased number of c-fos positive cellswere found in the 1H-N, 8H-N and 24H-N groups compared to the NC group(p<0.01). A significant difference was also found between the 8H-N and8H-N/T groups (p<0.01), suggesting that treatment with a compositioncomprising HPN-07 and NAC down-regulates c-fos expression. in the DCN atthis time point after noise exposure.

FIG. 2 depicts the results of c-fos immunostaining density measurementin the PVCN. A significant increased number of c-fos positive cells werefound in the 1H-N group compared to the NC group (p<0.01), suggestingthat c-fos expression was up-regulated in the PVCN immediately afternoise exposure.

FIG. 3 provides examples of c-fos immunostaining images obtained fromthe anteroventral cochlear nucleus (AVCN) of the normal control (A), thenoise exposure (B, C, E) and the noise/treatment (D, F) groups by lightmicroscopy. Many positive c-fos stained cells were found in the AVCN 1Hafter noise exposure (B), suggesting that e-fos expression wasup-regulated in the AVCN immediately after noise exposure. The number ofpositive cell returned to the normal level 8H (C) and 7-21D (not shown)after noise exposure. However, there was a second peak of up-regulation24H after noise exposure. Positive stained cells in the DCN were countedand statistically analyzed (G). A significant increased number of c-fospositive cells were found 1H-N, and 24H-N groups compared to the NCgroup (p<0.05 or 0.01). A significant difference was also found betweenthe 8H-N and 8H-N/T groups (p<0.01), suggesting that administration ofthe composition comprising HPN-07+NAC down-regulated c-fos expression inthe DCN at this time point after noise exposure.

FIG. 4 provides a comparison of c-fos expression in the DCN, AVON andPVCN at different time points after noise exposure and HPN-07+NACtreatment. Significant differences were found among three nuclei in1H-N, 8H-N and 24H-N/T groups (p<0.05 or 0.01). The DCN hadsignificantly more c-fos positive cells than VCN at two time points (1Hand 8H) after noise exposure (p<0.01), suggesting the DCN is alsosensitive to noise exposure although it has direct auditory input onlyin its deep layer.

In sum, Example 1 demonstrates the following: (1) c-fos expression wasup-regulated in neurons of the cochlear nucleus immediately after noiseexposure; (2) c-fos expression returned to the normal level 24 hoursafter noise exposure in the DCN and 8H in the VCN; (3) a second peak ofup-regulation in the AVCN 24H after noise exposure; (4) more c-fospositive neurons were found in the DCN than in the VCN after noiseexposure; and (5) administration of a composition comprising HPN-07 andNAC can down-regulate c-fos expression in the DCN (8H) and the AVCN(24H). This data suggests that treatment with HPN-07 and NAC iseffective in reducing the effects of noise-induced traumatic braininjury in the DCN and AVCN. Furthermore, animals with psychophysicalevidence of noise-induced tinnitus have previously demonstrated elevatedspontaneous activity in the DCN, suggesting that such hyperactivity maybe related to the noise-induced tinnitus. Thus, these results alsosuggest that the combination of HPN-07 and NAC may be effective intreating noise-induced tinnitus and other conditions associated withnoise-induced injury to the cochlear nucleus.

Example 2

In the present example, cell type specific synapse activity markers,precerebellin, and neuron markers, PEP-19 (cartwheel cell marker) andNeuN, as well as transmission electron microscopic studies (TEM) havebeen used to examine synaptic degeneration in the DCN of chinchilla 10days after a 105 dB SPL octave-band noise exposure and in the absenceand presence of a composition comprising 4-hydroxy-α-phenyl butylnitrone, NAC and ALCAR (4-OHPBN+NAC+ALCAR) beginning 4 hours after noiseexposure.

Three to five year old chinchilla were divided into 3 groups (6 in eachgroup): 1) normal control; 2) noise exposure only (105 dB SPLoctave-band noise centered at 4 kHz for 6 hrs); 3) noise exposure plustreatment with 4-OHPBN+NAC+ALCAR beginning 4 hours after noise exposureand twice a day for the next 2 days. Animals in the treatment groupreceived 20 mg/kg of 4-OH-PBN dissolved in dimethyl sulfoxide (40%),polyethylene glycol 400 (40%), and saline (20%), 50 mg/kg of 20% NAC inwater (containing 0.05% edetate disodium, dehydrate, PH 7.0, HospiraInc., lake Forest, Ill.), and 20 mg/kg of ALCAR (Sigma-Aldrich Inc. St.Louis, Mo.) in saline. These agents were intraperitoneally administeredseparately. The brainstems were collected and fixed by 4%paraformaldehyde intracardial perfusion. The brainstems were seriallysectioned with a cryotome at a thickness of 18-20 μm.Immumohistochemical labeling using anti-precerebellin, anti-PEP-19, oranti-NeuN antibodies was carried out on the sections to evaluate theeffectiveness of the treatment on synaptses and neurons after noiseexposure. Immunodensity was measured as the number of immuno positivecells per square mm. Cell counts were analyzed statistically (onewayANOVA and Tukey HSD tests). Brain tissue from three chinchilla (one foreach group) perfused with 4% freshly depolymerized paraformaldehyde and0.125% glutaradehyde were used for TEM study to examine synapticdegeneration in the middle region of the DCN of chinchilla.

FIG. 5 provides examples of precerebellin immunostaining images obtainedfrom the middle region of the DCN of the normal control (5A), noiseexposure (5B) and the noise/treatment (5C) groups by light microscopy.Positive precerebellin stained cells were found in the fusiform soma anddeep layers of the DCN (arrows and arrowheads in A-C). Positive stainedcells in the fusifom soma layer were counted and statistically analyzed(D). Significant differences were found among the groups only in themiddle region of the DCN, but not in the lateral or medial regions. Inthe middle region, significant differences were found between the normalcontrol and the noise exposure groups (p<0.01); as well as between thenoise exposure and the noise/treatment groups (p<0.05).

FIG. 6 provides examples of PEP-19 immunostaining images obtained fromthe middle region of the DCN of the normal control (6A), the noiseexposure (6B) and the noise/treatment (6C) groups by light microscopy.Positive PEP-19 stained cells were found in the fusiform soma and deeplayers of the DCN (arrows and arrowheads in A-C). Positive stained cellsin the fusiform soma layer were counted and statistically analyzed (D).No significant differences were found among the groups in all regions(p>0.05).

FIG. 7 provides examples of NeuN immunostaining images obtained from themiddle region of the DCN of the normal control (7A), the noise exposure(7B) and the noise/treatment (7C) groups by light microscopy. Numerouspositive NeuN stained cells were found in the fusiform soma and deeplayers, and few in the molecular layer of the DCN. Positive stainedcells were counted and statistically analyzed (D). No significantdifferences are found among the groups in all regions (p>0.05).

FIG. 8 provides examples of nerve terminals surrounding cartwheel cellbodies (A-F) and their primary dendrites (D1, G-L) in the middle regionof the DCN of the normal control (left column), noise exposure (middlecolumn) and noise/treatment (right column) chinchilla. The images inpanels D-F and J-L are the higher magnification of the images in panelsA-C and G-I, respectively. Two types of terminals, PVD (pleomorphicvesicles, dense) and PVL (pleomorphic vesicles, lucent), were foundsurrounding cartwheel cell bodies and their primary dendrites. Noobvious changes were seen in the PVD synaptic terminals of all 3 groupsof chinchilla. Compared to the PVL of the normal control group, hugevesicles were seen in the PVL of the noise exposure and thenoise/treatment groups (arrowheads in E, F, K), but not in the PVL ofthe nerve terminals surrounding D1 of the noise/treatment group (L).Furthermore, the synaptic membranes which have a convex shape in thenormal control and noise/treatment groups (arrows in D, F, J and L)appeared flattened and with a lower density contour in the noiseexposure chinchilla (empty arrow in E and K).

In sum, example 2 demonstrates the following: (1) Down-regulatedexpression of precerebellin was found only in the middle region of theDCN of the noise exposure group; (2) noise exposure used in the presentstudy did not cause cartwheel cell loss (labeled by PEP-19) or otherneuron loss (labeled by NeuN) in the DCN; (3) TEM showed enlargedvesicles and flattened synaptic membranes in the nerve terminalssurrounding cartwheel cell bodies and their primary dendrites in themiddle region of the DCN; (4) synapse degeneration in the middle regionof the DCN is likely one of the results of noise exposure; and (5)administration of a composition comprising 4-OHPBN+NAC+-ALCARsignificantly restored precerebellin expression and decreaseddegeneration of synapses surrounding cartwheel cell body and theirprimary dendrites in the DCN. Thus, early treatment with4-OHPBN+NAC+ALCAR following noise exposure acts to preserve normalcentral auditory structure.

Example 3

The purpose of this example is to demonstrate the effect of2,4-disulfonyl PBN (HPN-07) in combination with NAC on various braininjury biomarkers in the hippocampus and cortex following blastexposure.

Male Long-Evans pigmented rats were exposed to 3 blasts (14 psi,1.5-minute intervals between blasts). Rats were injectedintraperitoneally with 300 mg/kg of NAC plus 300 mg/kg of HPN-07 innormal saline 1 hour after blast exposure and then continued twice a dayfor the following two days in the treatment group of rats. Rats with orwithout blast were injected carrier solutions and used as controls. Sixrats were used in each group and each time point. All animals wereintracardially perfused with 10% paraformaldehyde 3, 24 hours, 7 daysand 21 days after blast (6 rats at each time point in each group, total54 rats). Brains were collected and cryosectioned at 30 μm. Amyloidprecursor protein (APP) and Glial Fibrillary Acidic Protein (GFAP)levels were measured in the CA1 region of the hipoocampus and auditorycortex to indicate the level of trauma or blast induced brain injury.The levels were measured by immunohistochemical staining using rabbitanti-APP IgG (1:100, Millipore), or GFAP IgG (1:500, Millipore) andAvidin Biotin Complex (ABC) Method. Images were collected by light orconfocal microscopy. APP positive staining was counted and analyzedstatistically (ANOVA and post hoc tests) as demonstrated in FIG. 9.

APP staining is absent in the CA1 region of the hippocampus and cortexin the control group (no blast). However, 24 hours after exposure toblast, strong APP positive staining is found in the CA1 region ofhippocampus and the cortex indicative of brain injury. This injury isdecreased in both regions upon combination treatment with HPN-07 and NAC(confocal images not shown). It was also observed that APP proteinaccumulates in axons and cell bodies as a response to injury (confocalimages not shown). FIG. 9 provides a quantification of the APP positivebodies found in the confocal images. A significant difference was foundbetween normal control (NC) and blast groups (24H-B) (P<0.001 and 0.05),as well as between blast (24H-B) and blast plus treatment (24H-B/T) 24hours after blast (P<0.001). These results indicates that thecombination of HPN-07 and NAC one hour after blast exposure inhibits APPformation and expression in brain thereby suggesting that damageresulting from blast-induced brain injury is reduced with HPN-07 NACtreatment.

FIG. 10 depicts GFAP immunostaining images obtained from the dorsalcochlear nucleus (DCN-4×, FIG. 10A) and the middle region of the DCN ofthe normal control (20×, FIG. 10B), the blast exposure (FIG. 10C) andthe blast/treatment (FIG. 10D) groups by light microscopy 21 days afterblast, Positive GFAP stained cells are found in the all layers of theDCN while most of them are located in the superficial layer. Fewpositive cells are found in the normal control. More GFAP positive cellsare found in the DCN 21 days after blast and fewer positive cells arefound in the DCN after treatment. Positively stained cells in the DCNwere counted and statistically analyzed, and the results are provided inFIG. 10E. Significant differences were found between the groups in themiddle region of the DCN (p<0.05), but not in the lateral and medialregions of the DCN (all p>0.05). These results demonstrate thattreatment with HPN-07 and NAC may inhibit glial activity after blastexposure with a regional effect in the DCN. This data supports thatNAC/HPN-07 treatment reduces blast-induced TBI, including auditorycenters such as the DCN and auditory cortex.

Example 4

The purpose of this example is to investigate the effect of HPN-07 andNAC on blood-brain barrier permeability

Male Long-Evans pigmented rats (with body weights between 360 and 400 g,Harlan Laboratories, Indianapolis, Ind.) were injected intraperitoneallywith a combination of 300 mg/kg of NAC plus 300 mg/kg of HPN-07, whichwere both dissolved in 5 ml/kg of physiological saline solution. Animalsin the control group were injected i.p. with a similar volume of saline.Drug or saline was administered once on the first day and then twice aday for the following two days.

Seven days after the initial drug administration magnetic resonanceimaging (MRI) was used to detect the disruption of the blood-brainbarrier (BBB). Contrast enhancement noted on post-contrast T1-weightedMRI after injection of contrast agent (Magnevist=GdDTPA) is indicativeof blood-brain barrier disruption. The hyperintensity on the T1-weightedimages is resulting from leakage of the gadolinium based contrast agentinjected at the baseline.

Magnetic resonance images were obtained before and after injection of0.4 mmol Gd/kg of Magnevist and demonstrated areas of disruption of theblood-brain barrier. The MRI parameters and image scale factor were keptconstant for all T1-weighted images. The images were evaluated bothvisually and by region-of-interest (ROI) measurement. ROI measurementswere obtained at the time points (before and after injection of contrastmaterial on Ti-weighted images) brain. The difference in ROI signalintensity was normalized with respect to its value before injection ofcontrast material and reported as a % change as depicted in FIG. 11.

Since NAC is known to not increase blood-brain barrier permeability,these results were unexpected and indicate for the first time thatHPN-07 effectively increases blood-brain barrier permeability therebyleading to increased bioavailability of any co-administered compound.Thus, the current invention also relates to a method for increasingblood brain permeability, where the method comprises administering2,4-disulfonyl PBN to a patient in an amount sufficient to increase thepermeability of the blood brain barrier and administering a secondcompound or substance, wherein the second compound or substance is beingused to treat or diagnose a condition of the brain.

Example 5

The purpose of this example is to determine the long-term effects of2,4-disulfonyl PBN in treating injury to the hippocampus and entorhinalcortex resulting from noise-induced TBI.

In this Example, three-5 year old chinchilla were freedom divided into 3groups (6 chinchilla at each time point in each group). Chinchilla innoise exposure and noise plus treatment groups were exposed to 105 dBSPL Octave hand noise centered at 4 kHz for 6 hours. Chinchilla in thenoise plus treatment group were received HPN-07 treatment (300 mg/kg,i.p) started 4 hours after noise exposure, and then twice a day for thefollowing 2 days. Chinchilla in the normal control and the noise exposedonly groups were received carrier solutions (i.p.). Chinchilla wereeuthanized and perfused with 4% paraformaldehyde in PBS 21 days and 6months after noise exposure. Brains were dissected out and post-fixed inthe fixative for one week. The brains were Cryosected at 30 μm. Goatanti-doublecortin (1:100) was used to label neural precursor cells.Images were taken from CA1 regions of hippocampus and the entorhinalcortex with a light microscope. Doublecortin staining reflectsneurogenerosis in the brain after neuron damage and death after noiseexposure.

FIG. 12 represents doublecortin immunostained sections of thehippocampus in control subjects not exposed to noise (FIG. 12A),subjects 6 months post noise exposure without treatment (FIG. 12B) andWith HPN-07 treatment (FIG. 12C). As demonstrated therein, the densityof doublecortin staining is noticeably increased in the subjects exposedto noise compared to subjects treated with HPN-07 which display adoublecortin density comparable to the control subjects.

Similar results were obtained in the entorhinal cortex as depicted inFIG. 13. FIG. 13A shows very low levels of doublecortin staining insubjects under control conditions (not exposed to traumatic noise).FIGS. 13B and 13D demonstrates increased doublecortin staining 21 daysand 6 months, respectively, following exposure to a traumatic noiseevent. FIGS. 13C and 13E demonstrate that treatment with HPN-07decreases the level of doublecortin staining at both time points.

Taken together, these results suggest that noise exposure resulted inincreased stem cell/repair activity in the hippocampus and entorhinalcortex that persists long after the traumatic event. Furthermore, thedecreased repair activity in the subjects treated with HPN-07 suggeststhat this compound is effective to reduce the tissue injury caused bythe traumatic noise event. The hippocampus plays important roles in theconsolidation of information from short-term memory to long-term memoryand spatial navigation, and the entorhinal cortex functions as a networkhub for memory and navigation and also serves as the primary interfacebetween the hippocampus and neocortex. Thus, these results also suggestthat HPN-07 may be effective to treat noise induced long-term memory(LTM) deficits.

The examples demonstrates the effectiveness of 2,4-disulfonyl PBN, aloneand in combination with NAC in treating noise-induced and blast-inducedbrain injury. In particular, the use of 2,4-disulfonyl PBN has beenshown to reduce the cellular and molecular effects associated withsecondary conditions resulting from traumatic brain injury. Finally,2,4-disulfonyl PBN unexpectedly increases blood brain permeability whichleads to increased bioavailability of the compound.

As used herein, a “pharmaceutically effective amount” is an amount of apharmaceutical compound or composition having a therapeutically relevanteffect on cellulalar damage or function, tissue damage or function, orother functional or physical symptoms resulting from traumatic braininjury including, but not limited to noise-induced tinnitus. Atherapeutically relevant effect relates to some improvement in thephysical or functional symptoms of traumatic brain injury or a change inthe cellular, physiological, anatomical or biochemical markersassociated with traumautic brain injury, including noise-induced andblast-induced brain injuries. In compositions comprising the combinationof 2,4-disulfonyl PBN and NAC or 4-OHPBN and NAC and ALCAR, apharmaceutically effective amount may be a dosage which ispharmaceutically effective for each compound, or in dosages which aresub-clinical for each compound, i.e., less than pharmaceuticallyeffective for each individually, or a combination thereof, provided thatthe combined dosages are pharmaceutically effective.

In one embodiment, a method for treating traumatic brain injury ortinnitus comprises administering to an organism a pharmaceuticallyeffective amount of a composition comprising 2,4-disulfonyl PBN and NAC.In one aspect, the composition comprises at least two parts NAC forevery part of 2,4-disulfonyl PBN, i.e. a ratio of 2:1 to 2.5:1, NAC to2,4-disulfonyl PBN. In another aspect, the composition comprises equalparts of 2,4-disulfonyl PBN and NAC. Furthermore, the concentration ofNAC used in the composition of NAC with 2,4-disulfonyl PBN may besubstantially less than treatment of a patient with NAC alone. Thecompositions may comprise between about 70 mg and about 1200 mg of2,4-disulfonyl PBN and from about 700 mg and about 4000 mg of NAC.Furthermore, compositions comprising 2,4-disulfonyl PBN may beadministered at a dose of between about 1 mg/kg to about 400 mg/kg bodyweight and more likely around 300 mg/kg body weight. Compositionscomprising NAC may be administered at a dose of between about 5 mg/kg toabout 300 mg/kg body weight. These ranges are based on the examplesincluded herein and do not limit the range of pharmaceutically effectiveamounts for other organisms.

In another embodiment, a method for treating traumatic brain injury ortinnitus comprises administering to an organism a pharmaceuticallyeffective amount of a composition comprising 4-OHPBN+NAC+ALCAR. Suchcomposition can have a dose range between about 5 mg/kg and about 300mg/kg for NAC, between about 5 mg/kg and about 150 mg/kg for 4-OHPBN andbetween about 5 mg/kg and about 500 mg/kg for ALCAR when ALCAR, NAC and4-OHPBN are used in combination.

One skilled in the art from a reading of this disclosure will likelyrecognize related compounds which will also provide satisfactoryresults. Further, although the foregoing examples treated the testsubjects one to four hours following noise exposure and blast exposure,treatments administered within shorter time periods should be aseffective and will likely be preferred. In addition, treatmentsadministered longer than 48 hour post noise exposure, blast exposure,stress or other cause of traumatic brain injury may also be effective.As such the foregoing disclosure is merely considered to be exemplary ofthe current invention with the true scope of the current invention beingdefined by the claims.

The invention claimed is:
 1. A method for treating noise-induced orblast-induced injury to cochlear nucleus, comprising administering to apatient in need thereof a pharmaceutically effective amount of acomposition comprising 2,4-disulfonyl a-phenyl tertiary butyl nitroneand N-acetylcysteine.
 2. The method of claim 1, wherein the compositionis administered orally.
 3. The method of claim 1, wherein thecomposition is administered intravenously.
 4. The method of claim 1,wherein the injury to cochlear nucleus is induced by exposure to noise.5. The method of claim 1, wherein the injury to cochlear nucleus isinduced by exposure to blast.
 6. The method of claim 4, wherein thecomposition is administered between one to four hours after the exposureto noise.
 7. The method of claim 5, wherein the composition isadministered between one to four hours after the exposure to blast. 8.The method of claim 1, wherein the composition further comprises one ormore compounds selected from the group consisting of Acetyl-L-Carnitine,glutathione monoethylester, ebselen, D-methionine, carbamathione andSzeto-Schiller peptides.
 9. The method of claim 1, wherein thecomposition consists essentially of2,4-disulfonyl a-phenyl tertiarybutyl nitrone and N-acetylcysteine as active ingredients.
 10. The methodof claim 1, wherein the composition down-regulates c-fos expression indorsal cochlear nucleus of the patient.
 11. A method for treatingnoise-induced or blast-induced injury to cochlear nucleus, comprisingadministering to a patient in need thereof a pharmaceutically effectiveamount of a composition comprising 2,4-disulfonyl a-phenyl tertiarybutyl nitrone.
 12. The method of claim 11, wherein the composition isadministered orally.
 13. The method of claim 11, wherein the compositionis administered intravenously.
 14. The method of claim 11, wherein theinjury to cochlear nucleus is induced by exposure to noise.
 15. Themethod of claim 11, wherein the injury to cochlear nucleus is induced byexposure to blast.
 16. The method of claim 14, wherein the compositionis administered between one to four hours after the exposure to noise.17. The method of claim 15, wherein the composition is administeredbetween one to four hours after the exposure to blast.
 18. The method ofclaim 11, wherein the composition down-regulates c-fos expression indorsal cochlear nucleus of the patient.